A potentiostat is an essential piece of analytical equipment for studying electrochemical devices and reactions. As the design of electrochemical devices evolve, applications for systems with multiple working electrodes have become more common. These applications drive a need for low-cost multi-channel potentiostat systems. We have developed a portable, low-cost and scalable system with a modular design that can support 8 to 64 channels at a cost as low as $8 per channel. This design can replace the functionality of commercial potentiostats which cost upwards of $10k for certain applications. Each channel in the multi-channel potentiostat has an independent adjustable voltage source with a built-in ammeter and switch, making the device flexible for various configurations. The multi-channel potentiostat is designed for low current applications (nA range), but its purpose can change by varying its shunt resistor value. The system can either function as a standalone device or remotely controlled. We demonstrate the functionality of this system for the control of a 24-channel bioelectronic ion pump for open- and closed- loop control of pH. In the last few decades, the use of miniaturized electrochemical devices has grown rapidly and found diverse applications in scientific and consumer products  and a custom Hardware Attached on Top (HAT) printed circuit board (PCB) that provides an interface between the SBC and the stackable boards, as well as supplying the necessary power. In this paper, the SBC is a low-cost Raspberry Pi 3 Model B+ computer that has Wi-Fi, Ethernet, 40-GPIO connectivity, and runs Raspbian OS version 10 (Buster) and Python 3.5. The multi-channel potentiostat has two operation modes, Standalone (that operate the multi-channel potentiostat independently) and Application Program Interface (API) mode . The HAT is designed in the form-factor of a Raspberry Pi and can accommodate up to eight stackable boards. The HAT contains +5, -5, +3.3, +2.8, and -1.65 V voltage regulators, TCA9548A, I2C multiplexer, DS3231 real-time clock, and a backup battery. It can interface with a Raspberry Pi via GPIO pin headers, one I2C communication bus, and one shift register bus.Each stackable board contains eight analog potentiostats with adjustable voltage sources, built-in ammeters, analog switches, and digital communication buses to the base unit. Each board is powered by a six-pin stacking header and interfaces with a Base unit via one of a 4-pin I2C cables. A Shift register is either chained with a custom-designed HAT or with another stackable board’s port. The most basic configuration consists of one base unit and one stackable board, but the system has a maximum of eight stackable boards for each single base unit. As a result, the multi-channel potentiostat support ranges from a minimum of 8 channels to a maximum of 64 channels.μA.Potentiostats designed for low current micro-electrode electrochemistry application in both aqueous and non-aqueous solutions need a wider voltage range (>±2 V) and a narrow current measurement range (<± 10 uA) . TherefoVo = 2.42 * Vin − 4. OPA209 is chosen due to it low power, low noise characteristics, and has a high short-circuit current rating. The MCP4728 has a resolution of 12 bits together with the OPA209 level-shifter result in an output resolution of 1.95 mVThe potentiostat circuit is split into two main stages: output and input. The output stage consists of three components: a Digital-to-Analog Converter (DAC), a level-shifter, and an analog switch. The DAC used in the proposed design is Microchip Technology MCP4728, which is a quad buffered 12-bit integrated circuit with a built-in high precision internal voltage reference, programmable I2C address, and EEPROM as nonvolatile memory. The DAC interfaces with the base unit via I2C allow up to four MCP4728 to connect and supports various I2C speeds such as Standard (100 kbps), Fast (400 kbps), and High Speed (HS) Mode (3.4 Mbps). The DAC output is between 0—3.3 V. However, the desired output range is ± 4 V. The level-shifter is a non-inverted Texas Instruments OPA209, which has Op-amp gain of 2.42 and a 2.8 V bias voltage. It is used to shift and scale the signal from 0–3.3 V to ± 4 V. The shifted and scaled output can be calculated as One feature that distinguishes the multi-channel potentiostat from other potentiostat designs to date is the ability to disconnect (open-circuit) individual output channels. The analog switches are added to connect or disconnect individual electrodes, altering electrode configurations automatically. Maxim Integrated MAX326 is a quad CMOS Analog switch, Single Pole Single Throw (SPST), and Ultra-Low-Leakage. It allows the multi-channel potentiostat to connect or disconnect to the electrode independently. The analog switches have 10 pA maximum leak current and 2 pC typical charge injection. In a low current application, leak current from a switch could unintentionally actuate the electrochemical device making these ultra-low leak characteristics crucial.MΩ) to measure nA range currents.The input stage of the multi-channel potentiostat measures the current from an adjustable voltage source that consists of an ammeter and Analog-to-Digital Converter (ADC). Unlike traditional potentiostat designs that use a transimpedance amplifier for current measurements, our multi-channel potentiostat has a shunt resistor. This approach might lower the accuracy but it allows simultaneous recording of multiple voltage sources. For instance, the multi-channel potentiostat with eight channels can operate four independent, two-electrode systems simultaneously, where four standard potentiostats would be required for the same setup. Additionally, the benefits of the multi-channel potentiostat are unlike traditional potentiostats because it can interface with electrochemical cells with three or more electrodes. The system is designed for high impedance load , is used to mitigate measurement errors due to the bias current. The shunt resistor value is 10k 0.1%, and the instrumental amplifier gain is 100. Consequently, the current to voltage conversion ratio is 1 nA to 1 mV. This design measures the current range from -1,650 to +1,650 nA. The current measurement range is adjusted by changing the value of the gain resistor for the instrumental amplifier, or shunt resistor, or both. Due to the limited range of the ADC, the instrumental amplifier output (-1.65 to +1.65 V) is shifted to match the 0—3.3 V of the ADC input. The output from the instrumental amplifier is fed into the inverted op-amp with an active low-pass filter to minimize noise and level-shifted by 1.65V. The inverted op-amp output is passed through another passive first-order low-pass filter with a cut-off frequency of 72 Hz to reduce the high-frequency noise. The OPA4209 is chosen as an op-amp for both input stages where it offers low voltage noise, quiescent current, offset voltage, and a short-circuit current of 65 mA. ADS1115 is a four single-ended or two differential inputs 16-bit ADC I2C interface with built-in programmable gain (PGA), low-drift voltage reference, and four pin-selectable addresses. This design uses the ADS1115 as four single-ended inputs with a maximum sampling rate of 860 samples per second. As a result, the input stage ranges from -1,650 to +1,650 nA with resolution of 125 uV or 0.125 nA.The standalone software consists of two main stages: initialization and execution. During the initialization stage, the script initializes all hardware components such as the DAC, ADC, shift-register, and real-time clock. Then, the calibration process starts by switching off analog switches (open-circuit), measuring ADC values during the no-load condition, and averaging the recordings to store as a baseline. After the calibration process, the program shows all predefined protocols that are stored in CSV format and prompt a user input to select which protocol to execute. Once the protocol is selected, the program will run until a user terminates it. While in the execution stage, the software displays real-time current measurement values. At the same time, it logs all of the states of the device, such as a timestamp, voltage output of different channels, and current measurement data in CSV format.In addition to the predefined protocols that run in Standalone mode, the multi-channel potentiostat offers an API where a user can control the multi-channel potentiostat remotely. The multi-channel potentiostat also connects with an external controller via an Ethernet or Wi-Fi connection. Similar to the standalone version, the API has the same initialization process. However, instead of executing a predefined protocol in the execution stage, it is listening for a User Datagram Protocol (UDP) command. The API allows users to set the voltage output of the multi-channel potentiostat and get current measurement feedback. Similar to standalone-mode, the multi-channel potentiostat states and output are also logged in CSV file format. API wrappers are available in both Python3 and Matlab libraries.The PCBs are designed on a standard 1.6 mm FR4 two-layer PCB with a dimension of 72.5 x 57 mm (Base) and 71.7 x 57 mm (Stackable). Both PCBs are manufactured by PCBWay (China); the cost, including shipping is approximately $ 2 per board. All electronic components are purchased from Digi-Key Electronics . The PCBs are populated and assembled at the University of California at Santa Cruz, shown in As an initial validation of the proposed device, we tested the linearity of the input and output signals. We also tested the performance of 8-channel multi-channel potentiostat under realistic load conditions. MΩ resistor and sweeps between two voltages to generate a variety of current conditions. A digital multimeter is used to measure the output of an instrumental amplifier and level-shifting op-amp. Similarly, the output test results show the error from linearity (between the current and multi-channel potentiostat’s ADC input) is less than 1% of the full-scale.MΩ resistor to simulate a high-impedance load. The multi-channel potentiostat’s output is programmed to sweep between -1.65 and +1.65 V, and the current is measured using the ADC. It is shown that the current varies linearly with the voltage (between -1.65 μA to +1.65 μA). As additional validation of the proposed system, we calculated the load’s resistance as 1.00969 MΩ, compared to 0.997 MΩ when measured with a digital multimeter.μm × 250 μm) and an Ag/AgCl pellet counter electrode (2 mm dia. × 2 mm thick) in 50 mM NaCl solution (pH 7). The palladium nanoparticle coating is chosen due to palladium’s ability to reversibly form palladium hydride by absorbing hydrogen (H) when negatively biased in the presence of water [The multi-channel potentiostat’s performance during sensitive cyclic-voltammetry (CV) experiments is validated against a commercial system (Autolab PGSTAT128N). For both systems, we run linear sweep voltammetry tests between -0.9V and 0.5V. of water . The expThis is explained by the combination of environmental noise and error from the multi-channel potentiostat’s output stages. The environment noise can be minimized by better shielding of the potentiostat which includes cables and an electrochemical cell. The output stage of the multi-channel potentiostat can be improved with changing a 12-bit DAC to a DAC with higher resolution such as 16-bit and adding an additional calibration step.Amperometry is a technique used to study redox reactions by providing a DC voltage between two electrodes and measuring the generated current. Ion pumps are bioelectronic devices that can manipulate physiological processes in vitro and in vivo delivering ions and small molecules directly to living matters , 16. TheIn many applications , insteadμA.We have proposed a low-cost, multi-channel portable electrochemical array controller that allows users to conduct experiments with complex electrochemical cells (more than three-electrodes) in aqueous and non-aqueous solutions. The proposed design is modular: it is possible to scale up to 64 parallel potentiostats with adjustable voltage sources ± 4 V and maximum currents ± 1.5 The device is validated in a cyclic-voltammetry experiment against a commercial system (Autolab PGSTAT128N). To demonstrate the functionality of the multi-channel potentiostat device, we used it to control an ion pump that had one AgCl electrode in a NaCl solution and nine palladium individual target micro-electrodes. The amperometry results show good agreement with the actuation voltage and the fluorescent results.The device could be remotely controlled via API enabling it to integrate with other laboratory systems. Here we had shown that it successfully integrated in an ion pump closed-loop controlled to monitor the changes into the ion concentration through imaging it using a fluorescent microscope.Although the bench-top potentiostat would perform more proficiently in similar conditions, the multi-channel potentiostat is an ideal solution in scenarios that are not feasible for bench-top potentiostats, and where a high degree of accuracy is not required . In conclusion, we have proven that the proposed device is scalable, portable, significantly cheaper than commercially available systems with similar characteristics, and can be controlled remotely. Therefore it could be used as a component in lab settings that need to be operated remotely.S1 File(ZIP)Click here for additional data file.


Caenorhabditis elegans. However, the response to even higher doses is not clear. First, to investigate the effects of high-dose radiation on the locomotion of C. elegans, we investigated the dose range that reduces whole-body locomotion or leads to death. Irradiation was performed in the range of 0–6 kGy. In the crawling analysis, motility decreased after irradiation in a dose-dependent manner. Exposure to 6 kGy of radiation affected crawling on agar immediately and caused the complete loss of motility. Both γ-rays and carbon-ion beams significantly reduced crawling motility at 3 kGy. Next, swimming in buffer was measured as a motility index to assess the response over time after irradiation and motility similarly decreased. However, swimming partially recovered 6 h after irradiation with 3 kGy of γ-rays. To examine the possibility of a recovery mechanism, in situ GFP reporter assay of the autophagy-related gene lgg-1 was performed. The fluorescence intensity was stronger in the anterior half of the body 7 h after irradiation with 3 kGy of γ-rays. GFP::LGG-1 induction was observed in the pharynx, neurons along the body, and the intestine. Furthermore, worms were exposed to region-specific radiation with carbon-ion microbeams and the trajectory of crawling was measured by image processing. Motility was lower after anterior-half body irradiation than after posterior-half body irradiation. This further supported that the anterior half of the body is important in the locomotory response to radiation.Radiation damages many cellular components and disrupts cellular functions, and was previously reported to impair locomotion in the model organism  Adineta vaga (tetraploid rotifer) is 1200 Gy  was used  C. elegans strains ) at a dose of 0–6 kGy. Irradiation was carried out at the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) facility at QST-Takasaki. The LET of the carbon-ion broad beam passing through the worm was approximately 119 keV/μm at the surface of the NGM plate. For all irradiation experiments, non-irradiated control worms were handled in parallel with irradiated worms.Whole-body irradiation with γ-rays to d method ,19. Bried method ,29. BrieTo evaluate the effects of whole-body irradiation with γ-rays or carbon-ion beams on motility, immediately after irradiation, crawling, such as forward motion, backward motion, and turning of the worms on the NGM plates spread with a bacterial lawn was video-recorded A. A digiC. elegans-specific image processing software, WormLab®4.0   strain was purchased from the Caenorhabditis Genetic Center. After irradiation, adult DA2123 worms were incubated on an NGM plate spread with a bacterial lawn for 0–28 h at 20 °C. After incubation, worms were paralyzed using 0.5 M sodium azide and mounted on an agar pad. Then, GFP fluorescence from the worms was observed by confocal microscopy  using a 488 nm argon laser. To identify the expression site, we conducted in the least fluorescent setting.To detect the induction of autophagy, the DA2123 [12C5+) particles were accelerated by an AVF cyclotron at TIARA and with the method previously described [To investigate the involvement of specific regions of the body, we employed the targeted-microbeam irradiation technique. Carbon-ion  was approximately 106 keV/μm and that at the bottom of the microfluidic channel (depth from the surface of a sample = 195 μm) was approximately 110 keV/μm. Therefore, the track-averaged LET of carbon ions passing through a worm enclosed in a microchannel was approximately 108 keV/μm. From this value we calculated the number of ions that would give the desired absorbed dose to the targeted region. As a practical case, 600,000 particles correspond to a dose of 3 kGy at a micro-aperture area of Φ60 μm.w/v) gelatine, 1 mM MgSO4, 1 mM CaCl2, and 5 mM potassium phosphate, pH 6.0), and transferred one by one to fresh NGM plates with OP50 using a platina picker . Thereafter, we conducted the same crawling assay described above in the case of whole-body irradiation. For all irradiation experiments, non-irradiated control worms were handled in parallel with irradiated worms.In order to target an area the same size as the body width of an adult worm, we used a Φ60-μm micro-aperture (beam exit)  for antet test. p < 0.05 was considered significant. All statistical analyses were performed using Microsoft Excel software .Statistical analysis was performed on more than 3 independent experiments of whole-body irradiation. The significance of differences was examined using the Student’s C. elegans was reduced by whole-body irradiation with high-dose γ-rays and carbon-ion beams. The decreased motility after whole-body irradiation partially recovered. Anterior half-body irradiation by carbon-ion microbeams decreased motility. Autophagy was induced during the recovery of decreased motility after whole-body irradiation (This study showed that motility in adult adiation . This st


The incidence of hand foot and mouth disease (HFMD) has increased in recent years, making it a very common childhood illness worldwide. The relationship between different enterovirus genotypes and disease severity is not clearly understood. Given that enteroviruses are transmitted through the gastrointestinal tract, we hypothesized that variation in intestinal microorganisms of the host might play a role in the prognosis of HFMD.We carried out a meta-transcriptomic-wide association study of fecal samples obtained from a cohort of children  with mild and severe HFMD and healthy controls.Clostridium sp. L2-50 and Bacteroides stercoris ATCC 43183 were enriched in the guts of children with severe HFMD and KEGG enrichment was found between mild and severe cases.We found there was no significant difference in the amount of each virus type between the mild and severe cases. Genes of enterovirus 71 (EV71) and coxsackievirus A (CV-A) from the severe and mild cases did not show significant clustering. Bacteroides and Clostridium may be used as predictive markers for a more efficient prognosis and intervention. The enrichment of intestinal bacteria genes with functions may facilitate the development of severe symptoms for HFMD patients.Intestinal microorganisms appear to interact with enterovirus to determine the progression of HFMD. Genes of The online version contains supplementary material available at 10.1186/s12879-021-06748-7. Hand foot and mouth disease, caused by various enteroviruses, is a common disease in children worldwide. Enterovirus 71 and coxsackievirus A16 (CV-A16) were the most common HFMD-causing pathogens reported; however, since 2010, coxsackievirus A6 (CV-A6) emerged as the most common serotype in Shenzhen, China . It evenAlthough enteroviruses are the pathogens causing HFMD in children, they also belong to common intestinal microorganisms. Therefore, bacterial colonization in the intestine may be associated with emergent disease . AlternaTo evaluate the potential association between the intestinal microbial community and disease severity of HFMD and identify novel predictive clinical biomarkers for severe cases, we performed total-RNA-seq shotgun sequencing on fecal samples obtained from individuals with HFMD, and compared the intestinal microorganisms between mild and severe cases and identified enrichment of specific bacteria in severe cases. Accordingly, we built an index model for predicting whether an individual will develop a severe case of HFMD based on intestinal microbiota composition. This study should enhance our understanding of HFMD beyond the current focus on enteroviruses and are expected to serve as guidance for developing new clinical treatments.This study was conducted at Shenzhen Third People’s Hospital and approved by the Ethics Committees of Shenzhen Third People’s Hospital. Informed consent was collected from all of our patients by their parent or legal guardian.All the samples were collected from pediatric patients at Shenzhen Third People’s Hospital and Guangzhou Women and Children’s Medical Center, and were classified into 4 groups: healthy (H), mild case (M), severe case (S), and severe case after one week of treatment (A). The distinction for classifying a case as mild or severe was made according to the World Health Organization’s Diagnostic Guide of Hand Foot and Mouth Disease . SpecifiIn total, 297 fecal samples were collected from patients with suspected HFMD who visited the Shenzhen Third People’s Hospital and Guangzhou Women and Children’s Medical Center as well as from volunteers visiting for physical examinations. The mean age of all individuals was 25.5 months (range 5–104 months); 184 were male and 113 were female. All samples were maintained at − 80 °C and shipped on dry ice before sample processing.3 feces sample in 1 mL phosphate-buffered saline. The RNA was finally eluted with 60 μL of Nuclease-free Water using the QIAamp Viral RNA Mini Kit  [Total fecal RNA was extracted from the feces supernatants that were obtained from dissolving a 0.5-cmGermany) , accordiAbout 2 μg of total RNA was fragmented with Covaris E210. Using these short fragments as templates, random hexamer primers were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized in the reaction buffer containing dNTPs, RNase H, and DNA polymerase I. Short double-stranded cDNA fragments were purified with a QIA Quick PCR extraction kit (QIAGEN), eluted with the Elution Buffer, and then end-repairing was performed with the addition of 3′-A overhangs. Next, the short DNA fragments were ligated to Ion Torrent-compatible barcoded adapters. DNA fragments of a selected size 200 bp) were gel-purified and amplified by polymerase chain reaction (PCR). AMPure beads (Beckman Coulter) were used to purify the resulting library, and an Agilent 2100 BioAnalyzer (Agilent Technologies) and Agilent BioAnalyzer DNA High-Sensitivity LabChip (Agilent Technologies) were used to determine the concentration and size of the library [0 bp wereThe raw reads were mapped to the human genome (hg19) using TMAP , and unmapped reads were first mapped to the enterovirus reference sequences  through TMAP  and then filtered . Reads mapped to the enterovirus reference sequences were clustered, and then highly similar sequences were removed using CD-HIT   was calculated after 100 random samplings with replacement.X” refers to the expression of the genes selected from mRMR selection and a indicates the redundancy coefficient of each gene.In order to identify microorganism genes that could be used as potential markers for distinguishing between severe and mild cases of HFMD, all of the differentially expressed genes (DEGs) between any two groups were determined using the Wilcoxon rank-sum test (p < 0.01). The minimum redundancy–maximum relevance (mRMR) feature selection method described by Peng  was usedA meta-transcript linkage analysis modified from a metagenomic linkage group (MLG) analysis  was carrA Wilcoxon rank-sum test was used to identify potential genetic markers distinguishing the different groups according to HFMD severity. The candidate genes were annotated to the Integrated Reference Genome of the Human Gut Microbiome (IGC) Kyoto Encyclopedia of Genes and Genomes (KEGG) database . The perq).In our multiple hypothesis test, we used the q-value to measure the false-positive rate  . Based ogiven by :\documenC is the number of reads uniquely classified to microbe species, N is the number of reads uniquely classified to all microbe species, and L is the genome length of the classified species. A Wilcoxon rank-sum test was used to identify whether viruses other than enterovirus differed or showed enrichment between mild and severe cases. In order to confirm the right result, all the clean Reads belonging to virus (including herpesviruses) were checked through an independent alignment by BLAST to NT database. The power analysis was done used software G*Power3.1.9.7, the result sample size should more than 73 with the actual power 0.85.All of the virus, bacteria, and fungi complete genome sequences were collected from the NCBI ftp site, and all sequences were merged for constructing a microbiome database using Kraken ; in addiOf the 254 patients (group M and S), 227 (89.4%) were tested positive for enterovirus, including 16 (6.3%) patient co-infection with 2 kinds enterovirus. Enterovirus positive was detected from about 92.0% (92/100) of the severe cases and 53.3% (16/30) even after treatment. The co-infection rate was 6.3% (16/254) overall, and was 7% and 5.8% in severe and mild cases, respectively, with no significant difference . And 14 different virus genotypes were identified with different detection rates: EV71 , CV-A4 , CV-A16 , CV-A10 , CV-A6 , CV-B5 , CV-A2 , CV-A5 , CV-A8 , CV-A24 , HEV9 , HEV13 , HEV1 , and EV96 . EV71 infection was predominant among the HFMD cases .We further evaluated all of the differences in viruses between mild and severe cases to evaluate the contribution of virus type to disease severity. We found that several typical plant viruses differed between mild and severe cases Table . These pFor each sample, a sufficient number of sequences should be sequenced to prepare for effective comparison among groups Fig. A. The nuFirmicutes, Bacteroides, Prevotella, Desulfovibrio, Mitsuokella, and Blautia, whereas more than half of these genes were unknown  in order to build a similar model to distinguish between mild and severe cases  that were all less than 24 months of age to evaluate the microorganism enrichment tendency according to disease status, irrespective of age effects Fig. . For theBacteroides (37.97%) followed by Clostridium (12.66%), as described above  and conducted a cluster analysis between the groups and species Fig. A. The H idium 12.6%, as deEscherichia coli ABU 83972, Bacteroides fragilis 3_1_12, Ruminococcaceae bacterium D16, Clostridium methylpentosum DSM 5476, and Coprobacillus sp. D7. The highest abundance of these bacteria was found in the S group, followed by the M group, with the lowest abundance in the A group. This suggests that a higher abundance of these bacteria may contribute to increasing the disease severity, and that the abundance would decline after treatment.There were 5 kind of bacteria that showed a significant difference between the M and S group enriched in S, the S and A group enriched in S, and the A group and M group enriched in M  in all samples Fig. C: EscherBacteroides, Blautia, Ruminococcus, Clostridium, Faecalibacterium, Alistipes, and Pseudoflavonifractor  detected  in more than 30% of all samples of both severe and mild cases. However, there were few differences in the microorganisms between these CV-A-positive groups. Therefore, all of the CV-A-positive samples were pooled as one group and compared to the EV71-positive samples as another group, and similar enrichment analysis was conducted. Both the gene and species diversity were significantly different between the CV-A group and EV71 group . The diversity of the EV71 group was slightly higher than that of the CV-A group  and conducted a cluster analysis between the groups and the functions Fig. . The M aThe positive identification of enterovirus serves as an important indicator for a clinical diagnosis of HFMD. Most clinical diagnoses are based on one of three types of detection by RT-PCR: EV71-positive, CV-A16-positive, and universal enterovirus-positive. Therefore, RT-PCR is only capable of detecting one type of co-infection: EV71 plus CV-A16. However, EV71 may co-infect with many other enteroviruses, especially coxsackievirus, as detected in the present sample  to find microorganisms gene marker index distinguishing Severe & Mild cases. A: minimum redundancy–maximum relevance (mRMR) feature selection method and leave-one-out cross-validation (LOOCV) steps. ‘x’ means any genes and ‘a’ means weight of the gene in the formula, it can be plus and minus. B: Find the optimum lowest error rate subset to build a linear discrimination classifier. Fig. S5. The rpkm value of all 20 gene markers for the mild and severe case distinguish index model. Yellow, mild case, Red, severe case. The annotation of gene markers were list left together with the weight of each gene in the mRMR selection method. The red box highlight the shared 2 genes in the model build from samples only EV71 positive. Fig. S6. EV71 positive samples index model for distinguish severe and mild case. A: leave-one-out cross-validation (LOOCV) to find the optimum subset to build a linear discrimination classifier . B: For each individual, an index was calculated to evaluate the risk of severe HFMD disease. The histogram shows the distribution of indices for all individuals. Red means mild case and blue means severe case. C: new samples for test use the index model (Verify: M:S = 10:10). Lowest error rate: 16.67%. Mild case and severe case were distinguished by the index model (p < 0.05). D: Venn chart for the two index model. ev + means the model was built from the samples with all enterovirus positive and ev71 + mean the index model was built from the samples with only enterovirus 71 positive. Fig. S7. Intestinal microorganisms difference between EV71 and CV-A groups. A: gene profile between CV-A and EV71 group. B: species profile between CV-A and EV71 group. . C: MLG species enrichment in EV71 group. The size of the circle indicates abundance of the MLG. The color of the circle indicates their taxonomic assignment. Connecting lines represent Spearman correlation coefficient values above 0.6 (grey) and below − 0.6 (blue).


Ornithodoros but not hard ticks. However, the underlying mechanisms evolved in the vectorial capacity of soft ticks are not well-understood. Here, we found that a defensin-like peptide toxin OPTX-1 identified from Ornithodoros papillipes inhibits the enzyme activity of the ASFV pS273R protease with a iK=0.821±0.526μM and shows inhibitory activity on the replication of ASFV. The analogs of OPTX-1 from hard ticks show more inhibitory efficient on pS273R protease. Considering that ticks are blood-sucking animals, we tested the effects of OPTX-1 and its analogs on the coagulation system. At last, top 3D structures represented surface analyses of the binding sites of pS273R with different inhibitors that were obtained by molecular docking based on known structural information. In summary, our study provides evidence that different inhibitory efficiencies between soft tick-derived OPTX-1 and hard tick-derived defensin-like peptides may determine the vector and reservoir competence of ticks.African swine fever virus (ASFV) is a large double-stranded DNA virus and causes high mortality in swine. ASFV can be transmitted by biological vectors, including soft ticks in genus  Ornithodoros soft ticks (Ornithodoros ticks for ASFV (African swine fever virus (ASFV), which causes a highly contagious and hemorrhagic disease of swine with a 100% mortality rate, is the only known DNA arbovirus and can be transmitted by ft ticks . As a veft ticks . The virfor ASFV . Howeverfor ASFV . So far,Mesobuthus martensii Karsch has been reported to inhibit hepatitis B virus replication  found in vertebrates, invertebrates, and plants. They are important endogenous antimicrobial factors to combat invading pathogens and have therapeutic potential for infectious disease . For exalication . An1a, aand ZIKV . Severaland ZIKV .Ornithodoros papillipes inhibits the enzyme activity of the pS273R protease with a iK=0.821±0.526μM. Interestingly, we also found that the analogs of OPTX-1 from hard ticks show more inhibitory efficiency on pS273R protease. The molecular docking results show that OPTX-1 interacts with pS273R mainly through the active site in the core domain. Given the lack of effective treatments against ASFV, our results may also provide insights into anti-ASFV drugs development from natural origin.As a large DNA virus, ASFV capsid comprises 8,280 major capsid protein p72 and 60 penton protein copies . It is k2 at 37°C.PAM cells were prepared from a healthy 2-month-old pig . Lungs w2 at 37°C.Vero cells  were obtained from Kunming Cell Bank, Kunming Institute of Zoology, Chinese Academy of Science. Vero cells were cultured in DMEM medium  supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, and 100μg/ml streptomycin in 5% COEscherichia coli strain BL21(DE3)  and overexpressed. Protein expression was induced by 0.5mM IPTG  and further purified on a Ni-NTA column. SDS-PAGE analysis revealed >98% purity of the final purified recombinant protein.The codon-optimized cDNA of ASFV pS273R protease was synthesized  as previously reported . The ful18 RP-HPLC using a linear acetonitrile gradient, and the purity was detected by MALDI-TOF-MS (Peptides were synthesized by GL Biochem (Shanghai) Ltd.  and analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry to confirm their purity greater than 98%. The linear reduced peptide was dissolved in 0.1M Tris·HCl and 0.1M sodium chloride buffer (pH 8.0) at a final concentration of 30μM glutathione containing 0.1mM reduced glutathione and 0.5mM oxidized glutathione at 25°C for 24h. Oxidized and folded peptides were fractionated by analytical CI-TOF-MS .To test if the peptides inhibited pS273R protease activity, assays were conducted in 96-well black microplates utilizing Bz-Nle-Gly-Gly-Arg-AMC  as a substrate . MonitoriK was determined according to the reported methods.The effects of peptides on thrombin  were determined as described previously . SubstraActb gene  was used as a reference gene for relative quantification.For virus infection, PAM cells were infected with ASFV SY18 strain (GenBank: MH766894) at 0.1 MOI with or without OPTX-1 administration for 48h in a biosafety facility. The total DNA were extracted with TIANamp Genomic DNA Kits . qRT-PCR was performed on the StepOnePlus Real-Time PCR Systems , and the primer sequences are p72-F (TTAGGTACTGTAACGCAGCA) and p72-R  was added to the plates, and the clotting time was recorded. Clotting time was calculated by measuring the time to half maximal increase in absorbance. All the analyses were monitored by a microplate spectrophotometer  at the absorbance of 650nm.Blood coagulation assays were performed according to our previous study . HealthyCell viability was evaluated by conventional 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assays in 96-well plates. After a 24h treatment by testing sample, MTT was added to each well to a final concentration of 0.5mg/ml and incubated at 37°C for 4h. The MTT solution was then removed, and dimethyl sulfoxide (DMSO) was added to solubilize the MTT-formazan crystals in living cells. The absorbance of the resulting solution was measured at 570nm.The Modeller v9.18 software was used to construct a model of three tick defensins, OPTX-1, persulcatusin, and longicin by homology modeling and then used ZDOCK v3.0.2 software to perform molecular docking .t test. Value of p≤0.05 was considered significant.Figures were generated using the GraphPad Prism 6 software . Data are given as mean±SEM. Statistical analysis was performed using two-tailed Student 18 RP-HPLC using a linear acetonitrile gradient, and the purity was further confirmed by MALDI-TOF-MS  worldwide. It is reasonable to speculate that the tick-derived peptides possess anticoagulant activity. As shown in ASF currently has no effective pharmacological treatment. As competitive inhibitors of pS273R protease of ASFV, OPTX-1 and its analogs are expected to provide peptide precursor molecules for the development of therapeutic drugs for ASF, for further evaluation, cytotoxicity was assessed by using the MTT test. As shown in Ornithodoros soft ticks. We assumed that the vectorial efficiencies of ticks are depended on the successful but limited replication of ASFV.ASFV affects domestic and wild members of the Suidae family, leading to a wide range of symptoms from chronic or persistent infection to acute hemorrhagic fever, and inflicts up to 100% mortality . The maiin vitro.As a crucial SUMO-1-specific protease of ASFV, pS273R represents an attractive therapeutic target . This stThe docking model indicated that the active site of pS273R core domain directly interacts with the peptides mainly through the hydrogen bonds. The cysteine protease activity of pS273R can be abolished by mutation of the predicted catalytic histidine and cysteine residues or inhibited by sulfhydryl-blocking reagents . HoweverOrnithodoros soft ticks can be orally infected, maintained and transmitted ASFV vertically  among ticks, and horizontally to naive pigs. Several studies emphasized that vector competence was not only related to the tick but also to the ASFV strain. Thus, it is necessary to test the inhibitory effects on pS273R protease as well as the virus replications of tick defensins with different ASFV variants.Defensins are known major immunoregulatory components of ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites . FunctioThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JW, MJ, BY, AL, ZJ, TZ, and YL conducted the experiments. RL and LJ designed the experiments and provided guidance for the research. LJ analyzed the data and wrote the paper. PK revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the African Swine Fever Research Emergency program of the Chinese Academy of Sciences (CAS) (KJZD-SW-L06-02). LJ is partly supported by the National Natural Science Foundation of China (NSFC) grant (31900331 and 32070444), Science and Technology Department of Yunnan Province (202001AW070019), Chinese Academy of Sciences “Light of West China” program and Youth Innovation Promotion Association (2019378). RL is partly supported by the NSFC grant (3193001521761142002), the CAS grants , and Yunnan Province grant .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


During the COVID-19 pandemic, SARS-CoV-2 variants have emerged and spread worldwide. The Delta (B.1.617.2) variant was first reported in India in October 2020 and was classified as a “variant of concern (VOC)” by the WHO on 11 May, 2021. Compared to the wild-type strain, several studies have shown that the Delta variant is more transmissible and has higher viral loads in infected samples. COVID-19 patients infected with the Delta variant have a higher risk of hospitalization, intensive care unit (ICU) admission, and mortality. The Delta variant is becoming the dominant strain in many countries around the world. This review summarizes and analyses the biological characteristics of key amino acid mutations, the epidemic characteristics, and the immune escape of the Delta variant. We hope to provide scientific reference for the monitoring and prevention measures of the SARS-CoV-2 Delta variant and the development strategy of a second-generation vaccine. As ofhttps://www.gisaid.org/), and Pango are currently in use by scientists. The SARS-CoV-2 variants were classified as “variant of concern (VOCs)” and “Variant of Interest, VOI)” by the WHO. At present, Alpha B.1.1.7   into glycine (Gly) at site 614 of the spike protein, was first discovered in the UK and quickly became the dominant epidemic strain in the world, attracting widespread attention , 8. The 2012/01) , Beta B.2012/01) , Gamma P2012/01)  and Delt2012/01) –15 and i2012/01) –18. The SARS-CoV-2 invades host cells by binding the spike protein to angiotensin-converting enzyme-2 (ACE2) –21. The The L452R mutation is located in the receptor-binding motif (RBM) region in the RBD region, containing residues that bind to ACE2 –28. AnalWilhelm A et al.  found thin vitro cell culture studies have shown that the Delta variant carrying T478K is more likely to undergo secondary mutation in a low titer antibody environment, leading to the failure of host antibody immunization (Compared with the other two B.1.617 lineages (B.1.617.1 and B.1.617.3), Delta (B.1.617.2) does not have the E484Q mutation but has a unique T478K mutation . An in snization .In vitro, cell culture experiments revealed that the size of floating syncytia in the D614G/P681R mutant-infected culture was significantly larger than that in the D614G mutant-infected cell culture , and the cleavage of this region is the key to host cell entry . Several culture . These dIn addition, the neutralization analyses of pseudoviruses showed that three monoclonal antibodies against RBD had 1.5-fold (1.2 ~2.65) decreased neutralization activity by against pseudoviruses with the D614G/P681R mutation. The neutralizing activity assay using the 19 sera elicited by the BNT162b2 vaccine (two doses) showed that pseudoviruses carrying the D614G/P681R mutation are significantly resistant to the vaccine-induced NAbs compared to the D614G pseudoviruses . These rStefano Pascarella et al.  reportedThe above studies suggested that L452R, T478K, and P681R are the three key mutations of the SARS-CoV-2 Delta variant. These mutations increased transmissibility and generated immune escape of the Delta variant, as shown in vs. 5.7). It has been reported that the fifth generation of cases emerged just ten days after the first case was infected with the Delta variant  after infection with the Delta variant was 2-3 days, which was shorter than that of the wild-type strain (3-7 days). The basic reproductive number  of the Delta variant (R0:4.04~5.0) was higher than that of the wild-type strain (R0:2.2~3.77). In addition, the generation time  was 2.9 days (95% CI: 2.4–3.3), which was much shorter than the wild-type strain (2.9  variant –43.vs. 19.05%)  were 1260-fold higher than those of the wild-type strain  when PCR was first used to detect SARS-CoV-2. Moreover, the number of patients infected with the Delta variant containing the viral loads > 6x105 copies/mL in oropharyngeal swabs was significantly higher than that of the wild-type strain when the viruses were first detected (80.65%  19.05%) . In addi 19.05%) .Moreover, epidemiological studies from Guangzhou also showed that patients infected with the Delta variant could spread in a short time even if individuals did not converse when sharing toilets or eat in the same space –48. SimiThese data indicated that the Delta variant has higher transmissibility and a shorter incubation period. Patients infected with the Delta variant had higher viral loads .Delta (B.1.617.2) was first reported in India in October 2020 . Ram VS According to nationwide sampling conducted by the genomics company Helix in San Mateo, California, Delta is rising fast, while Alpha fell from more than 70% of cases in late April to around 42% by mid-June 2021 . Since mThe first local infection of the Delta variant was identified in Guangzhou, Guangdong Province, China, on 21 May 2021 . From 21Many clinical studies have reported that critical COVID-19 illness caused by infection with the wild-type strain includes acute respiratory distress syndrome; coagulopathies ; septic It has been reported that the neutralization activity of 30% (6/20) of mAbs against the Delta variant was reduced more than 5-fold compared with that of the wild-type strain. In addition, the neutralization activity of 45% and 5% of convalescent plasma against Delta was reduced by approximately 3-10-fold and >10-fold, respectively , 66. IntIn Israel, two doses of the Pfizer vaccine (mRNA vaccine) can reduce symptomatic infections by 94%, related hospitalization by 87%, severe cases by 92%, and the risk of Delta infection by 79% , 67. In The Delta variant can cause breakthrough infections in vaccinated populations in Guangzhou. The effectiveness evaluation of inactivated-virus vaccines-Sinovac/CoronaVac against the Delta variant during the epidemic in Guangdong showed that the efficacy of Sinovac/CoronaVac to prevent close contact infection was 69%, the efficacy of Sinovac/CoronaVac to prevent the development of symptomatic COVID-19 was 73%, and the efficacy of Sinovac/CoronaVac to prevent severe COVID-19 cases was 95% , 71.All of these data revealed that, although the Delta variant presents partial immune escape, the mRNA vaccine (AstraZeneca and Pfizer vaccine) and inactivated-virus vaccines (Sinovac/CoronaVac) still have a protective effect against the Delta variant.The severity of COVID-19 is mainly related to hosting factors, especially cellular immune responses in patients. Patients with mild COVID-19 and recovered patients with severe COVID-19 exhibit a normal humoral and a T cell-mediated immune response to effectively eliminate the virus . VaccineThis review describes the biological characteristics of the L452R, T478K, and P681R mutations of the Delta variant spike protein. These mutations impact Delta variant biological behavior, including increased transmissibility and immune evasion. COVID-19 patients infected with Delta have a higher risk of hospitalization and ICU admission than patients infected with other VOCs  and wild-type strains. The Delta variant may be the most transmissible VOC and is becoming the main epidemic variant in many countries worldwide.DT conceived and wrote the manuscript and prepared figures. YS and JZ contributed to the data collection and prepared the table. QY conceived and contributed to the modification and revision of the manuscript. All authors contributed to this article and approved the submitted versions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We study the existence of nontrivial solution branches of three-coupled Gross–Pitaevskii equations (CGPEs), which are used as the mathematical model for rotating spin-1 Bose–Einstein condensates (BEC). The Lyapunov–Schmidt reduction is exploited to test the branching of nontrivial solution curves from the trivial one in some neighborhoods of bifurcation points. A multilevel continuation method is proposed for computing the ground state solution of rotating spin-1 BEC. By properly choosing the constraint conditions associated with the components of the parameter variable, the proposed algorithm can effectively compute the ground states of spin-1  Comprehensive investigation of spinor and rotating BEC can be found in the review articles5 and the book6, respectively. Recent investigation on this topic can be found, e.g., in11. More precisely, Nolan et al.7 presented a model of a spin-squeezed rotation sensor utilizing the Sagnac effect in a spin-1 BEC in a ring trap. Gautam and Adhikari9 exploited variational method and numerical solution to study the vortex-bright solitons in a quasi-two-dimensional spin-orbit-coupled hyperfine spin-1 BEC using the mean-field theory. Eto et al.10 studied the dynamics of BEC of 14 (CGPEs) as follows:Experimental results on rotating spinor Bose–Einstein condensates (BEC)t the time variable, m the atomic mass, x-, y- and z-direction, respectively, and s-wave scattering lengths for the channel of total hyperfine spin jz-component of the angular momentum. Moreover, the superscript “16, in which a dissipative GPE for the condensate wave function is coupled with a heat base described by the Boltzmann equation. Another simpler model is the so-called stochastic projected Gross–Pitaevskii equation (SPGPE), in which thermal fluctuations of the bosonic field are taken into account at stochastic forcing19.Here 22.For completeness we define the state of lowest energy of a BEC system with fixed number of particles as the ground state. The states with energies greater than the ground-state energy are called excited states. The linear Zeeman (LZ) energy and the quadratic Zeeman (QZ) energy are given by33. In particular, the preconditioned imaginary time evolution method (PITEM), or the so-called continuous normalized gradient flow (CNGF) was widely used. See35. Recently, the performance of PITEM and continuation methods on some test problems in boson-fermion mixtures was compared in36. Published articles on numerical study of spin-1 BEC is also abundant. See e.g.,40. Research papers on numerical investigation of rotating spin-1 BEC can be found e.g., in47.Various numerical methods have been proposed for computing the ground state solution of both one- and two-component rotating BECz-direction, i.e., z-direction. We integrate the energy functional over z. Then from Eq.  and Moreover, the Fréchet derivative ting Eq.  into Eq.ting Eq. , we obtaboth Eq.  and 18)\documentIn the first three cases, Eq.  reduces We have the following result.Ifthen Eq. (reduces to a single GPE.then Eq.  reduces From Eqs.  and 9)  9) we hafrom Eq.  we have  and Eq.  reduces L is a positive constant yet to be specified which is large enough. Next, we transform the domain 62 to discretize Eq.  BECn of Eq. . It suffn of Eq. . Startin See Eq. . A detaiTheoretically we can use both the chemical potential ~ in Eq.  for somet of Eq.  can be es of Eq.  can be es of Eq. .To begin with, we consider the first level continuation algorithm with e of Eq.  is a nonWe denote a solution curve of Eq.  by\documc. Thus a unit tangent vector 67 and the further references cited therein. To compute the ground state solution, we start with an initial point s, we obtain68 and the further reference cited therein for details.Assume that a parametrization via arc-length is available on the solution curve with Eq. . Differe39 for detailed discussions. Now we rewrite the magnetization , a, a\docum of Eqs.  are the The algorithm described above may be briefly summarized as follows.A multi-level continuation method for rapidly rotating spin-1 BEC.Initialization:a given small positive number for implementing Level 2.initial particle number used in Levels 1 and 2.given.Level 1.SetandImplement the first level continuation algorithm to trace the ground state solution untilLevel 2.SetImplement the second level continuation algorithm under the constraintuntil the normalizationis reached.Level 3.SetImplement the third level continuation algorithm under the constraintStop the curve-tracing whenis reached.40.Table For tracing the ground state solution curve of ultrarapidly rotating spin-1 BEC, we use the angular velocity ~ in Eq.  for some69 for 70 for 71 for 73, we have In this section we report the implementation results of Algorithm 74, which are given bym and Order associated with the convergence behavior of (Convergence behavior of Algorithm entclass1pt{minimaentclass1pt{minima(i)19 but not exactly the same because of using different governing equations and coefficients.(ii)76. In addition, the two-norm of the third component 23. The result verifies the prediction numerically shown in Lemma M increases, the governing Eq. ((The ground state solutions of rotating spin-1 BEC for (i)19.(ii). Owing to the repulsive interspecies interaction, it is intuitional that vacancies like vortices in one component are filled by droplets in another in order to lowering the energy of the system. However, the spinor degrees of freedom can provide a platform to study topological quantum phenomena in such multi-component system. This kind of BEC are called spinor BEC. For a spinor M plays a key factor which makes Eq. (From Lemma akes Eq.  reduce t0.5, Eq.  almost d


Momordica charantia L., also named as bitter gourd or bitter melon in popular, is a common tropical vegetable that is traditionally used to reduce blood glucose. A peptide derived from bitter gourd, Momordica charantia insulin receptor binding peptid-19 (mcIRBP-19), had been demonstrated to possess an insulin-like effect in vitro and in the animal studies. However, the benefit of the mcIRBP-19-containing bitter gourd extracts (mcIRBP-19-BGE) for lowering blood glucose levels in humans is unknown.The fruits of This aim of this study was to evaluate the hypoglycemic efficacy of mcIRBP-19-BGE in subjects with type 2 diabetes who had taken antidiabetic medications but failed to achieve the treatment goal. Whether glucose lowering efficacy of mcIRBP-19-BGE could be demonstrated when the antidiabetic medications were ineffective was also studied.N = 20) and placebo group (N = 20), and were orally administered 600 mg/day investigational product or placebo for 3 months. Subjects whose hemoglobin A1c (HbA1c) continued declining before the trial initiation with the antidiabetic drugs were excluded from the subset analysis to further investigate the efficacy for those who failed to respond to the antidiabetic medications.Subjects were randomly assigned to two groups: mcIRBP-19-BGE treatment group (P = 0.057) and HbA1c (P = 0.060). The subgroup analysis (N = 29) showed that mcIRBP-19-BGE had a significant effect on reducing FBG  and HbA1c .The oral administration of mcIRBP-19-BGE decreased with a borderline significance at fasting blood glucose (FBG; All of these results demonstrate that mcIRBP-19-BGE possesses a hypoglycemic effect, and can have a significant reduction in FBG and HbA1c when the antidiabetic drugs are ineffective. Hypoglycemic efficacy of mcIRBP-19-BGE was evaluated in the type 2 diabetic patients.mcIRBP-19-BGE could significantly reduce FBG and HbA1c in the subjects who failed to respond to the antidiabetic drugs.The results demonstrated that mcIRBP-19-BGE had a hypoglycemic effect and could be an alternative treatment option for the patients when the antidiabetic drugs were ineffective.Today, diabetes mellitus (DM) has become a critical issue with about 425 million patients globally in 2017 within the age group of 20–79 years, according to the International Diabetes Federation . AlthougMomordica charantia L., popularly known as bitter gourd or bitter melon, is a common tropical vegetable that is traditionally being used to reduce blood glucose , had 68 amino acids, with a molecular weight of 7 KDa, and could bind with the insulin receptor (Momordica charantia insulin receptor binding peptid-19) and mcIRBP-9 (Momordica charantia insulin receptor binding peptid-9), respectively. Both peptides were able to bind with insulin receptor, activate the kinase activity and the downstream molecular communicators, and therefore, had the benefit of lowering blood glucose levels  for 1 month  for lowering blood glucose in diabetes. However, we could not recruit the type 2 diabetic patients without taking any hypoglycemic medication for ethical considerations. Thus, this human trial was designed to investigate the efficacy and safety of mcIRBP-19-containing bitter gourd extracts (mcIRBP-19-BGE) in type 2 diabetic patients who had failed to achieve the treatment goal under hypoglycemic medication treatment. We hypothesized that the investigational product possesses a hypoglycemic effect when the antidiabetic medications cannot achieve the treatment goal. Because this study adopted an add-on treatment design, the hypoglycemic efficacy of mcIRBP-19-BGE was further assessed in the subset subjects who showed no medication efficacy. The hypoglycemic efficacy was evaluated by fasting blood glucose (FBG) and HbA1c. In addition, several indicators of health conditions for the subjects were also evaluated.n = 21) and placebo group (starch: 600 mg/day) (n = 20). All study subjects underwent screening evaluation up to 7 days prior to administration of the investigational products. The privacy rights of the subjects have been well protected. Patients were also advised to take prescribed hypoglycemic medication under physicians’ instruction since the investigational products were not supposed to replace the regular treatment. In addition, all study subjects were asked to pay attention for better control of their blood glucose through the education in this study, and to maintain a stable diet and lifestyle.This randomized, double-blind, placebo-controlled, parallel comparison study was conducted in the Division of Endocrinology and Metabolism of Chung Shan Medical University Hospital (CSMUH) from May through November 2017. The protocol and study material were approved by the CSMUH Institutional Review Board (IRB), and were registered with the National Institutes of Health, ClinicalTrials.gov identifier: NCT03151837. The major enrollment criterion was that type 2 diabetes patients who had been treated with more than one oral medication but did not achieve the treatment targets (refers to steady dosage treatment for 3 months but still with FBG levels between 140 and 270 mg/dL and HbA1c 7–10%). Patients who had serum creatinine >1.8 mg/dL, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, or alkaline phosphatase >2.5 times of normal range, anemia , severe angina, moderate–severe heart failure with left ventricular hypertrophy, body mass index (BMI) lower than 18 or greater than 38, sudden and recent changes in dietary habits (within 1 month) or weight change exceeding 10%, unstable medical condition or life expectancy of less than 6 months, a known history of allergy to ingredients in the test product, severe diabetes complications or acute disease deemed unsuitable for participation judged by the investigators, being pregnant or breastfeeding, or had been administered any experimental drugs within 30 days were excluded from this study. A total of 41 eligible subjects with written informed consent were randomly assigned to one of the two groups: mcIRBP-19-BGE capsules (600 mg/day)  were then used to assess the hypoglycemic effect of mcIRBP-19-BGE. The subset analysis also could be used to evaluate the hypoglycemic efficacy of mcIRBP-19-BGE under the ineffective treatment condition from the antidiabetic medications.®; batch number IN161116F01P) was obtained from Greenyn Biotechnology Co., Ltd . The investigational product was 100% made from fruits of Momordica charantia L. After washing and slicing, the fruits of Momordica charantia L. were extracted with 100% water. After filtration, centrifugation, concentration, freeze vacuum drying, pulverization, and sieving of the extracts, mcIRBP-19-BGE was prepared. The industrial product of mcIRBP-19-BGE contains approximately 0.17% of mcIRBP-19. The product has also been tested to contain undetectable levels of heavy metals, plasticizers, or pesticides. Both of the products used in this trial, mcIRBP-19-BGE (300 mg/capsule) and placebo starch (300 mg/capsule), were identical in appearance and were manufactured by ISO certified United Biocaps Corp. . The study subjects were suggested to take one capsule before lunch and dinner each day, and not to take within 30 min of other medications. The dosage (i.e. 600 mg/day of mcIRBP-19-BGE) was recommended by the manufacturing company, and it contained approximately 471 nmol of mcIRBP-19 . According to the previous study , along with health condition indicators  were measured and tested at baseline and 3 months after the initiation of study treatment. All laboratory evaluations were performed by the Department of Laboratory Medicine of CSMUH.Compliance rate was calculated using the formula: ‘the number of capsules taken by the subjects / the number of capsules should be taken by the subjects × 100%’.Subjective self-evaluation of negative effect after the administration of the study product was obtained at every doctor visit or through telephone interview to monitor the safety of the investigational product. Each patient was carefully monitored for the development of adverse events by the physician.All data of individuals who have completed the study were entered for efficacy and safety analysis. Data were presented as mean and standard deviation for continuous variables, and frequency and percentage were presented for categorical variables. The Mann–Whitney U test was performed to compare the change from baseline for study assessments between the two groups, and the Wilcoxon signed-rank test was used to compare the values before and after ingestion for each group. Categorical variables were compared between the two groups using Fisher’s exact test, and the changes before and after ingestion within each group were analyzed using the McNemar test. All statistical analyses were performed using SPSS version 18.0 for Windows , and the level of significance was set at 0.05.Among 42 subjects who have been enrolled into the study, one subject was excluded before randomization due to the treatment goal being reached during the screening period, and one subject from mcIRBP-19-BGE group dropped out because of the occurrence of papule all over the body . Of the The oral hypoglycemic drugs were classified as 1) insulin secretagogues (e.g. sulfonylurea [SU] and non-sulfonylurea [non-SU]), 2) insulin sensitizers , 3) α-glucosidase inhibitors, 4) dipeptidyl peptidase-4 (DPP-4) inhibitors, and 5) sodium glucose cotransporter 2 (SGLT2) inhibitors . Almost There were no significant differences in the baseline assessments, including glycemic indices, total protein, uric acid, liver and kidney function, vital signs, body weight, BMI, body fat, and circumferences of waist, hip, arm, and thigh between the two groups for both analysis sets . The comP = 0.057 and 0.060 for FBG and HbA1c, respectively) for the mcIRBP-19-BGE group (P > 0.05) were observed for the placebo group  for the mcIRBP-19-BGE group  were observed for the placebo group  was observed at month 3 for the placebo group (P = 0.044) for the mcIRBP-19-BGE group (P = 0.025) in arm circumference (P = 0.145) in arm circumferences for the mcIRBP-19-BGE group  and continued through 3 months  (P = 0.012); however, the change was with a borderline significance for subset individuals  , while n= 0.028) . However= 0.028)  and 4. TP = 0.500) after 3 months of administration for the placebo group in all subjects and from 0.0 to 13.3% (P = 0.500) in the subset, respectively (P = 0.063) in all subjects and from 0.0 to 28.6% (P = 0.125) in the subset, respectively  increased from 0.0 to 10.0% (ectively . For theectively . These rP = 0.051).The change from baseline for most of the evaluations was not significantly different between the two groups , 0.5–1.0% in nateglinide (a non-SU), 1.0–1.5% in metformin, 0.5–1.5% in TZD, 0.5–1.5% in α-glucosidase inhibitor, 0.4–1.1% in DPP-4 inhibitor, and 0.39–2.05% in SGLT2 inhibitor –26. The We wondered whether mcIRBP-19-BGE had a significant hypoglycemic effect on the type 2 diabetic patients who had failed to have the drug efficacy by the antidiabetic medications. According to the results from this study, mcIRBP-19-BGE at the used dosage (i.e. 600 mg/kg) could reduce blood glucose only for the subset patients who could not benefit from the hypoglycemic drugs. However, mcIRBP-19-BGE at the used dosage did not significantly decrease both HbA1c and FBG from the analysis of all subjects, suggesting that the hypoglycemic efficacy of mcIRBP-19-BGE was low for all of the type 2 diabetic patients who had taken antidiabetic medication but failed to achieve the treatment goal. However, the marginal hypoglycemic efficacy of mcIRBP-19-BGE for all subjects might be attributed to the interference from antidiabetic drugs. Because some subjects showed a steady decrease in HbA1c before the initiation of the study treatment (i.e. the drugs were still effective for these subjects), the hypoglycemic efficacy of mcIRBP-19-BGE may be interfered by the drug efficacy for both treatment and placebo groups at this situation. Another possible explanation for the marginal hypoglycemic efficacy of mcIRBP-19-BGE for all subjects might be due to the dose used. In this study, a dose of about 2.82 times that used in the previous animal study  may be iMomordica charantia relating to regulating heart rate in the literature. The regulating heart rate in subset patients but not in all study subjects implied that the efficacy of mcIRBP-19-BGE to regulate heart rate might be related to the hypoglycemic medications. The mechanism associated with the slowing in the heart rate by mcIRBP-19-BGE still needs further investigations. Moreover, the mechanism for the significant body weight decrease and the total protein increase after taking mcIRBP-19-BGE for 1 month for the subgroup analysis were not clear.Moreover, this study found that the diabetic patients had a decrease in the circumference of arms and thighs, which might be related to the increasing risk of sarcopenia from the diabetes . AlthougIn summary, this study has shown that mcIRBP-19-BGE can significantly reduce FBG and HbA1c levels for the patients who failed to respond to the antidiabetic medications. The hypoglycemic efficacy of mcIRBP-19-BGE approximately decreased HbA1c by 0.5%, on average, for a 3-month administration at a dose of 600 mg/day for the subset patients. All of these results have suggested that mcIRBP-19-BGE can be an alternative treatment option for the type 2 diabetic patients when the antidiabetic drugs are ineffective. The mcIRBP-19-BGE may also have the effect of preventing the decrease of the arm and thigh circumferences, and regulating the heart rate of the type 2 diabetic patients. Another clinical trial with a higher dose of mcIRBP-19-BGE is highly recommended for the further investigations.


Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to β-lactam antibiotics  by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes  were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to β-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to β-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.Avian pathogenic  Escherichia coli (APEC) causes avian colibacillosis, which results in serious losses to the poultry industry around the world [E. coli can cause serious growth defects [E. coli to antibiotics is related to AI-2. When the concentration of autoinducers reaches a threshold, population-wide changes in gene expression are triggered, resulting in the modulation of a variety of physiological characteristics, and changes in sensitivity to antibiotics [E. coli to β-lactam antibiotics by up-regulating the expression of TEM-type β-lactamase [pfs affects the sensitivity to antibiotics by affecting the synthesis of AI-2 has not been reported. In addition, Pfs has many functions, and it is not reported whether they are involved in bacterial resistance. Therefore, this study wants to study whether pfs affects the sensitivity of antibiotics through AI-2. This will help screen out important targets for antibacterial drugs.Poultry is one of the most widespread types of meat consumed worldwide. Poultry flocks are often raised under intensive conditions, using large amounts of antimicrobial agents to prevent and treat diseases, as well as promote growth. Antimicrobial resistant poultry pathogens may cause treatment failures, economic losses, and are also the source of resistant bacteria and genes (including zoonotic bacteria) that may pose a risk to human health . Avian phe world ,3. APEC he world . Quorum he world ,6,7. Thrhe world . AI-2 ishe world ,10. The he world . Pfs is he world . AI-2 bihe world . Both SAhe world . The lac defects . Previou defects . Howeveribiotics ,18. The actamase . As an iactamase . In prevactamase . Whetherpfs gene deletion strain (DE17Δpfs) and the pfs gene complementation strain (c-pfs) were constructed [The APEC strain DE17 was isolated from a sick duck with septicemia and neurological signs [structed . The bacE. coli ATCC 25922 was used as a quality-control strain. The sensitivity of APEC strains to antibiotics was tested via the agar diffusion method, using MH agar and commercially available discs (Oxoid), according to Clinical and Laboratory Standards Institute (CLSI) guidelines [The APEC strains DE17, DE17Δpfs and c-pfs were tested for susceptibility against 14 antimicrobial agents using the disc-diffusion method in agar MH, as described before . The folidelines .600 value of 1. The OD600 was adjusted to 0.5 with MH. Antibiotics are serially diluted two-fold in MH medium to make the ampicillin concentration range from 6.354–6250 μg/mL, the amoxicillin concentration from 0.049–500 μg/mL, the ceftazidime concentration from 1.1465–375 μg/mL, and the cefuroxime concentration from 0.244–250 μg/mL. The 96-well plates were prepared by adding 100 μL of MH broth to each well of the 96-well plate, and adding 100 μL of each of the four antibiotic solutions  to the first well. Then, 100 µL of each of the serial dilutions were transferred into 11 consecutive wells. Next, 95 µL of MH and 5 µL of inoculum were added to each well of the 96-well plate. The last well was used as a negative control, which contained 195 µL of MH without antibiotic and 5 µL of inoculum. The final volume in each well was 200 µL. The 96-well plates were incubated at 37 °C for 20–24 h. In addition, the molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by different species of bacteria and is the precursor of the signaling molecule AI-2. DPD can be used as exogenous AI-2, and different concentrations of DPD  were added to the culture medium to test whether AI-2 would affect the MIC of antibiotics against APEC.The MIC were determined as described previously . The bacg for 5 min. The cells were washed three times with PBS. According to the manufacturer’s protocol, the total RNA was extracted from the bacterial cell by the Trizol RNA isolation reagent . A Nanodrop spectrophotometer was used to evaluate the concentration and quality of RNA through the OD260/OD280 ratio. Then, the illumina HiSe-qTM2500/MiseqTM sequencing platform was used to construct the sequence libraries of DE17 and DE17Δpfs. The main differentially expressed genes (DEGs) were analyzed using gene ontology (GO) enrichment analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze the pathway analysis of DEGs. The Benjamini and Hochberg approaches were used to adjust the p-values, and p < 0.05 was set as the threshold for significant difference. The transcriptional levels of DE17 and DE17Δpfs cells were determined as described with some modification . When th600 value of 1.2, centrifuged at 1800× g for 5 min to collect the bacteria, washed with PBS 3 times, and the value of OD600 was adjusted to 0.5. Next, 1.92 mL bacterial solution of DE17, Δpfs, and c-pfs, respectively, were added into a quartz cuvette, and then 80 μL solution of NPN (1 mmol/L) was added to the mix. The fluorescence (the excitation wavelength of 350 nm and emission wavelength of 420 nm) was recorded with an F-4500 FL spectrophotometer . The value was recorded every 2 min until the number no longer changed, and the value was the highest fluorescence absorption value of the strain. The experiment was repeated three times. The permeability of the bacterial outer membrane was tested using the 1-N-phenylnaphthylamine (NPN) probe method, as described, with some modification . The ovetolC), and porin protein-encoding genes , and penicillin-binding protein genes (mrcA and ftsI). The primers for the target genes and the internal control gene dnaE were shown in 600 = 1.0), and total RNA was extracted by TRIzol reagent. DNaseI  was used to remove DNA from total RNA. The reverse transcription was performed using the PrimeScript 1 st Strand cDNA synthesis kit . The qPCR experiment was performed using the SYBR green PCR mix . The target genes were examined three times, and relative changes in gene expression levels were assessed using the 2−∆∆Ct method [The transcription level of the antibiotic resistance related genes was tested by a quantitative real-time PCR (qPCR) experiment. Those antibiotic resistance related genes include the selected efflux pump-encoding gene . Statistically significant differences between mean values were identified using the student′s pfs gene deletion, the inhibition zone of the deletion strain DE17Δpfs was larger than that of the wild-type strain and complement strain. The inhibition zone of the two antibiotics (amoxicillin and cefuroxime) against the DE17 was 15 mm, while the inhibition zone for the DE17Δpfs increased to 20 mm, indicating that pfs gene deletion was more susceptible to the cephalosporin antibiotics . Interestingly, after ibiotics .pfs gene. It is possible that pfs does not affect the sensitivity of antibiotics through the AI-2 pathway.The MIC of APEC was determined using four β-lactam antibiotics  were selected to determine the MIC of APEC. The results are shown in pfs deletion, including 346 global and overview maps genes , and the fluorescence absorption value of the DE17Δpfs strain was 1.6 times that of the complementation strain (p < 0.05). The results indicated that the outer membrane permeability of APEC was enhanced when the pfs gene was deleted.NPN is a highly susceptible fluorescent probe molecule, which is widely used to determine the permeability of the outer membrane of Gram-negative bacteria. The relative fluorescence absorption values of DE17, Δpfs, and c-pfs were detected using NPN at a final concentration of 30 μmol/L . The flupfs deletion (tolC and penicillin-binding protein genes (fstI and mrcA) transcription levels were significantly reduced after pfs deletion (p < 0.05). However, the transcription level of 3 porin protein-encoding genes  increased significantly (p < 0.05). Compared with the wild strain, the deletion strain was up-regulated 2.5-fold, 1.5-fold, and 3.0-fold, respectively. The transcription levels of these genes were restored in complementary strains. According to the analysis of the transcriptome, we used qPCR to detect the transcription level of β-lactam resistance related genes. The qPCR results revealed changes in the mRNA levels of β-lactam resistance-related genes in APEC following deletion . Comparepfs deletion, APEC was more susceptible to β-lactam antibiotics, especially cephalosporins antibiotics. Although the strain DE17Δpfs does not produce the signal molecule AI-2, and AI-2 has been reported to be associated with β -lactam antibiotic sensitivity, in this study, the addition of exogenous AI-2 did not make APEC resistant, so the sensitivity of strain DE17Δpfs to β-lactam antibiotics was not associated with the absence of AI-2 [E. coli  are responsible for the synthesis and assembly of the peptidoglycan sacculus that forms the rigid bacterial cell wall [In this study, it was found that, after  of AI-2 . All β-l of AI-2 . The pen of AI-2 . Acylate of AI-2 ,29. The ell wall . In thisell wall ,32. In fell wall . This isE. coli is a trimer. The three-dimensional structure shows a monomeric β-barrel composed of 16 anti-parallel β-strands containing pores [ompC, ompD, and ompF) was significantly increased in the strain DE17Δpfs in our study, which could explain the sensitivity to β-lactam antibiotics. When pfs is deleted, the expression of outer membrane protein increases, allowing antibiotics to enter the bacteria’s outer membrane and act on the cell wall more easily. Hence, it is easier to inhibit the growth of bacteria. The outer membrane permeability test of bacteria also verified this. The outer membrane permeability of the bacteria also becomes stronger in strain DE17Δpfs. The possible reason is that it is also caused by the increased expression of porin protein. In addition, we also examined the transcription level of the efflux pump gene. The outer membrane protein TolC is an envelope protein found in all Gram-negative bacteria, and it is essential for the excretion of excessive compounds [ompF, ompC, ompD), penicillin binding protein genes (fstI and mrcA), and efflux pump gene (tolC) (as shown in the The strategy of resistance to β-lactams, on the other hand, is to reduce drug concentration by increasing efflux, or in the case of gram-negative bacteria, by reducing penetration into the periplasm . The outng pores . Studiesng pores . Most β-ng pores . The traompounds ,41. The ompounds . The trapfs affects the expression of many metabolic genes. These metabolic genes may cause metabolic imbalances, which may be the main reason for the significant changes in antibiotic-related genes.In addition to participating in the synthesis of AI-2, Pfs also has many functions, such as methylation reactions, recovery pathways for adenine, and the synthesis of polyamines and vitamins. Transcriptome data proves that


P = 0.007) during the first 28 days and during the following 28–60 days  than those without infection, but after 60 days the additional risk of death was no longer significant .Persisting symptoms and dysfunction after SARS-CoV-2 infection have frequently been observed. However, information on the aftermath of COVID-19 is inadequate. We followed up people with severe mental illness (SMI) infected with SARS-CoV-2, and evaluated their longer-term mortality, using data from Cambridgeshire and Peterborough NHS Foundation Trust, UK. We examined the time course and duration of mortality risk from the point of diagnosis. After SARS-CoV-2 infection, people with SMI had a substantially higher risk of death (hazard ratio (HR) = 5.16, 95% confidence interval (CI) 1.56–17.03;  In this study, we conducted a longer-term follow-up of this group of patients and explored their longer-term mortality. We hypothesised that the mortality caused by SARS-CoV-2 infection beyond 28 days is still significant.People with severe mental illness (SMI) are at increased risk of being infected by SARS-CoV-2 and have increased mortality and a more severe course of COVID-19.9 SARS-CoV-2 status was confirmed by the reverse transcription polymerase chain reaction (RT-PCR) test recommended by the World Health Organization (WHO).We conducted a retrospective case-matched study using data from the mental health electronic clinical records of Cambridgeshire and Peterborough NHS Foundation Trust (CPFT) in the UK. CPFT covers a population of approximately 0.86 million people and provides secondary mental health and community physical health services. Data from CPFT include sociodemographic information, diagnosis, smoking status and death status. Clinicians enter this information during patients’ routine treatment in a systematic and standard way to ensure its accuracy.Data were collected from 1 March 2020 to 28 February 2021, to avoid the influence of the SARS-CoV-2 vaccination widely available from March 2021 in the UK. Eligible patients were those diagnosed with SMI. SMI was judged on the basis of the ICD-10 diagnosis codes F20–F29 , F30 (manic episode) and F31 (bipolar affective disorder). The exposure cohort were people with SMI who had laboratory-confirmed SARS-CoV-2. The control group were matched (at a ratio of 1:10) from the remaining people with SMI, matched on age (±1 year) and gender. The origin time was the date of COVID-19 diagnosis (exposure cohort) or the matched exposed case's date of COVID-19 diagnosis (control cohort). Follow-up was until the study end date, the patient’s final recorded CPFT data or the date of their death, whichever occurred first.The following sociodemographic variables were extracted: age at baseline (years), gender , marital status  and ethnicity (White versus other/not known). Smoking status was defined as being a past or current smoker. Death status for all patients known to the CPFT was extracted by weekly linkage to the national NHS Spine mortality data.10 as follows: myocardial infarction (ICD-10 codes I21–I25), hypertension , heart failure , diabetes mellitus (ICD-10 E08–E13), chronic kidney disease , obstructive lung disease  and cancer (ICD-10 C00–D49).We selected comorbidities that were plausible risk factors for a worse prognosisn (%). Differences between groups were tested via two-tailed t-tests (for continuous variables) and chi-squared tests .Continuous variables were reported as mean (s.d.) and categorical variables were reported as 6 and 60 days was derived from the plot  against follow-up time from the overall Cox regression, extracted from Schoenfeld residuals, a method originally used for graphically testing the proportional hazards assumption. We then conducted piecewise Cox regression to estimate the effect of SARS-CoV-2 infection on mortality during different follow-up periods. We chose 28 days and 60 days as the follow-up thresholds: 28 days is the limit of COVID-attributable death according to the National Health Service (NHS)the plot .Fig. 1AP < 0.05 was treated as statistical significance. We report results following the STROBE checklist.All analyses were done in R (v3.5.0) on Windows. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human participants/patients were approved by an NHS Research Ethics committee (reference 17/EE/0442). Individual patient consent was not necessary as data were extracted from de-identified electronic clinical records; no patients were involved in the development of the research question or the outcome measures, or in developing plans for the design and analysis of the study.https://doi.org/10.1192/bjo.2021.1046). The SARS-CoV-2 exposure cohort was followed up for an average of 159 days (s.d. = 123.6), significantly lower than that in the control cohort (186 days (s.d. = 120.1); P = 0.025). Some differences in baseline characteristics were identified between the two cohorts . Individuals in the SARS-CoV-2 cohort were more likely to be of ‘other/not known’ ethnicity (P = 0.003), to be former/current smokers (P = 0.001), and to have comorbid hypertension (P = 0.004), diabetes mellitus (P = 0.016), chronic kidney disease (P < 0.001) or cancer (P = 0.029). No significant difference was observed between the two cohorts for other characteristics, including age, gender, marital status, heart failure, myocardial infarction or obstructive lung disease. Sixteen deaths  were identified in the SARS-CoV-2 exposure cohort, a significantly higher fraction (P < 0.001) than in the unexposed cohort .Between 1 March 2020 and 28 February 2021, a total of 121 individuals with SMI and infected with SARS-CoV-2 were included in the exposure cohort, and 1210 individuals with SMI without SARS-CoV-2 infection were matched from 22 048 individuals with SMI and included in the control cohort  during the first 28 days, and this additional death risk reduced to 1.96-fold but was still significant  during the period of 28–60 follow-up days. After 60 days the additional risk of death following SARS-CoV-2 infection was not significant   .Results remained consistent after removing one patient with myocardial infarction and three patients with cancer.11 Our study has quantified the dynamic change of this association and reports on the aftermath of COVID-19 in people with SMI in a longer follow-up period. Our results have implications for clinical practice and suggest that people with SMI who are infected with SARS-CoV-2 should have follow-up observations for at least 60 days even if they have recovered or been discharged. Our findings also suggest that the 28-day cut-off adopted by the UK6 might underestimate COVID-19-associated mortality in this population.These findings supported our hypothesis that the additional mortality following SARS-CoV-2 infection persists beyond 28 days, although it decreases over time, and also indicated that this increased risk for mortality was no longer statistically significant about 60 days after infection. Our results before the 28-day point are in line with previous studies, which concluded that people with SMI have an increased risk for COVID-19-associated mortality.13 This study design provides a more accurate assessment of excess mortality related to COVID-19 infection in this population.To the best of our knowledge, this is the first study to examine mortality in people with SMI following COVID-19 infection using uninfected people with SMI as a comparison group. This is a strength of our study, as people with SMI are at increased risk of premature mortality due to a variety of causes. The crude death rate in our control group is 53.5 per 1000 persons per year, higher than that reported in general populations (10.4 per 1000 persons per year in 2020).14 Second, the sample is small, which prevented us from performing subgroup analyses for different types of SMI. Third, the control cohort may have included people with undiagnosed COVID-19 infection. Fourth, the use of secondary care data may have led to unmeasured biases . Fifth, the retrospective study design did not allow collection of data on persistent symptoms/disability among survivors of SARS-CoV-2 infection. Prospective studies in this area are required, considering also the specific cause of death. Similar studies in the general population are also needed, as a prolonged mortality risk may be a more widespread phenomenon.The current work has several limitations. First, our data did not contain information on the severity of symptoms for individuals infected with SARS-CoV-2. The long-term health sequelae are particularly relevant to those who required mechanical ventilation, for whom long-term complications and incomplete recovery would more likely be expected.